As to crops such as cereal crops and vegetables, F1 cultivars are being actively developed with features such as 1) an agricultural genetic character improved excellently by heterosis, 2) an equal quality of harvests, and 3) protectability of a breeder's right on the basis of segregation of genetic characters in the next generation. Actually, F1 varieties of many major crops have gone into actual use.
A method for seed product ion of an F1 cultivar is exemplified by cms/Rf seed production system comprising a cytoplasmic male sterile (cms) line and a line (hereafter may be abbreviated to Rf) for restoration from male sterility of the cultivar. For example, the method has been developed for cereals such as rice, Sorghum, and corn and an oil crop such as sunflower. These method have been developed by using a technique of breeding or cell fusion.
For Brassicaceae, the system for F1 seed production by applying self-incompatibility is widely applied. For rapeseed showing unstable self-incompatibility, however, the system for F1 seed production requires use of the cms line and the Rf line.
On the contrary, in recent years, a study has been conducted for using cytoplasmic male sterile line (Kosena cms) derived from Kosena radish and cytoplasmic male sterile line (Ogura cms) derived from Ogura radish for rapeseed. Both cms genes are encoded in a genome of mitochondria which is a cytoplasmic organelle, and their nucleotide sequences have been known. However, a molecular biological study using radish has not so developed and therefore, markers necessary for gene isolation have been seldom known. Thus, isolation of the gene from a nucleus is difficult. Therefore, introduction of the Rf gene has been achieved only for rapeseed by applying intergeneric crosses or cell fusion approaches to a radish line, of which fertility has been restored.
Furthermore, for Rf gene, 1 or more restorer genes are present according to each cms line of different plant species. For radish, the presence of Rf1 gene and Rf2 gene is necessary for restoration of fertility. In addition, it has been known that Rf1 gene reduces remarkably the accumulation of ORF 125 protein (M. Iwabuchi et al., Plant Mol. Biol. 39: 183-188. 1999) in mitochondria which is known as a cms-associated protein of radish (Jpn. J. Breeding 47 (separate volume 1): p. 186. 1997 and Jpn. J. Breeding 48 (separate volume 1): p. 197. 1998.)
In rapeseed, it has been also known by gene analysis study that radish Rf1 gene introduced by intrgeneric crosses or cell fusion reduces accumulation of ORF125 or ORF 138 protein (M. Grelon et al., Mol. Gen. Genet. 243: 540-547) which is known as the cms-associated protein, and that reduction of accumulation of these ORF 125 or ORF 138 protein coincides perfectly with fertility restoration phenomenon (N. Koizuka et al, Theor. Appl. Genet. 100: 949-955. 2000). The restoration of fertility of the male sterile line of rapeseed requires reduction of accumulation of the ORF 125 or ORF 138 protein. For this, Rf1 gene is an important gene.
However, concerning a nucleotide sequence of Rf genes, only Rf2 gene, which is a restorer gene for a T-cytoplasm which is one of maize cms, was identified and isolated. But no nucleotide sequence of Rf genes of other plant species has been known.